Engineered Environments For Examining Directed Cell Migration Tanea Hibler

Concordia International School Shanghai, Shanghai, China With the expertise of Dr. Ian Schneider from the Chemical and Biological Engineering Department at ISU

ABSTRACT

Dr. Ian Schneider is studying cancer cells, and how they make migrational decisions based on chemical and physical cues around them. His research group is studying this from several perspectives. Some of the research includes looking at how collagen and epidermal growth factor are involved in this process, they are tracking cell movements of rat cancer cells (MTLn3) with a high resolution video microscopy, and they are using microfluidic devices to view the movement of cells across a concentration gradient.

I have observed their research over the past 2 summers, learned some soft lithography techniques commonly used in biological engineering, how to culture and maintain MTLn3 cells, and I have created a 3D collagen chamber in which to view the cells. In addition, I have contributed to the analysis of the media type, protocols for making the slides, and the lab techniques that produce the best results for imaging these cells.

RESEARCH QUESTION/HYPOTHESIS Question: How does a 3D collagen environment impact how cancer cells migrate?

Hypothesis: Cancer cells will tend to move along aligned collagen fibers.

METHODS Before I had the opportunity to image any cells I had to learn the proper protocol for bringing cells up and culturing them, as well as those for passing cells from day to day. If I didn’t have happy cells then I had nothing to work with.

After learning protocols related to cell cultures, I had to experiment and come up with a protocol for mixing my collagen solution. Once I felt confident that I was making this solution correctly, and I had good images of my collagen fibers I was confident to add the cells . However, my cells kept dying. I tried keeping the solution off ice, on ice, putting on a heating plate immediately, etc, but the cells kept dying.

At this point it was suggested that I get a new media that would allow the cells to thrive outside of the incubator. I had to make the new media, bring it to the correct pH, and then start the experiment again. This was a trial and error process as I found out. The cell would die if they weren’t at the right temperature, or if the mixture was added in the incorrect order. I finally settled on the final procedure: •Put double sided tape on a slide •Cover it with a squeaky clean cover slip •Set this aside •Centrifuge cells, and prepare and mix with 200μL of 1X media •Cells remain in the incubator until they are needed •Mix 8μL of 10X media, 12.8μL of water, 2.0μL of NaOH, 58.2μL of collagen, and 20μL of 1X media + cells (everything but the cells are stored on ice) •Take 50μL of this mixture and pipette it onto the slide. This mixture should be pulled under the cover slip •Immediately put on dry bath and seal with valap and place in incubator •Image cells after a few hours

ACKNOWLEDGEMENT I would like to give a special thanks to EEOB for providing the funding for this program, and Adah Lesham-Ackerman for accepting me into the program and providing support during my time here.

I would also like to thank Dr. Ian Schneider for taking the time to accept a teacher into his lab, and taking the time to make this another relevant and meaningful experience for me.

And special thanks to Laura, Ariel, Nick, Amanda, Zach, and Alex who were always there to answer my questions.

RESULTS •The cells survived and I was able to image them overnight. •However, there is not a lot of movement during the imaging (the cells are not migrating across the collagen. Figure 1-3: Shows how the concentration of collagen affects the density of the mixture. Figure 4-5: Images of cells on different concentrations of collagen.

BACKGROUND Cancer impacts most people at some point in their lives, but tends to be more common as one ages, but even if we never get cancer, most of us have a family member, friend, co-worker, or maybe a neighbor that has been affected by it. Contributing to the research of how cancer cells make migrational decisions is important to me, as many people in my family have been affected by cancer, and it is worth while because it is something that will interest my students, but most of all it has allowed me to experience and observe a true research setting. In addition, helping scientists understand more about how cancer cells move may ultimately help doctors in grading the stage of the cancer.

DISCUSSION I hypothesized that the cells would move along the aligned collagen fibers. However, the group didn’t get to the point in the experiment where they could consistently align the collagen fibers, and we did not get a chance to experiment with different collagens, or try other procedures for making the 3D chamber. We did successfully make media in which the cells could survive, as well as successfully come up with the procedure for making the collagen mixture with the cells.

I expect that this project will continue in the future and someone will use a different technique in which cells are first plated on a cover slip, and then they migrate into a 3Dcollagen chamber.

EXTENSION The experience I have had in the lab has helped me to create a case study that I can use with my 9th grade Honors Biology students. In my case study students will be introduced to an undergraduate lab assistant who finds herself cutting corners to save time. This will allow me to discuss lab safety with my students, as well as expose them to cutting edge research.

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Ken Yamada’s Lab at NIH Centrifuging MTLn3 cells